Patients with ASPSCR1-TFE3 fusion achieve better response to ICI based combination therapy among TFE3-rearranged renal cell carcinoma

Background TFE3-rearranged renal cell carcinoma (TFE3-rRCC) is a rare but highly heterogeneous renal cell carcinoma (RCC) entity, of which the clinical treatment landscape is largely undefined. This study aims to evaluate and compare the efficacy of different systemic treatments and further explore the molecular correlates. Methods Thirty-eight patients with metastatic TFE3-rRCC were enrolled. Main outcomes included progression-free survival (PFS), overall survival, objective response rate (ORR) and disease control rate. RNA sequencing was performed on 32 tumors. Results Patients receiving first-line immune checkpoint inhibitor (ICI) based combination therapy achieved longer PFS than those treated without ICI (median PFS: 11.5 vs. 5.1 months, P = 0.098). After stratification of fusion partners, the superior efficacy of first-line ICI based combination therapy was predominantly observed in ASPSCR1-TFE3 rRCC (median PFS: not reached vs. 6.5 months, P = 0.01; ORR: 67.5% vs. 10.0%, P = 0.019), but almost not in non-ASPSCR1-TFE3 rRCC. Transcriptomic data revealed enrichment of ECM and collagen-related signaling in ASPSCR1-TFE3 rRCC, which might interfere with the potential efficacy of anti-angiogenic monotherapy. Whereas angiogenesis and immune activities were exclusively enriched in ASPSCR1-TFE3 rRCC and promised the better clinical outcomes with ICI plus tyrosine kinase inhibitor combination therapy. Conclusions The current study represents the largest cohort comparing treatment outcomes and investigating molecular correlates of metastatic TFE3-rRCC based on fusion partner stratification. ICI based combination therapy could serve as an effective first-line treatment option for metastatic ASPSCR1-TFE3 rRCC patients. Regarding with other fusion subtypes, further investigations should be performed to explore the molecular mechanisms to propose pointed therapeutic strategy accordingly. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-024-02044-5.

Patients with ASPSCR1-TFE3 fusion achieve better response to ICI based combination therapy among TFE3-rearranged renal cell carcinoma Introduction TFE3-rearranged renal cell carcinoma (TFE3-rRCC), characterized by Xp11.2 translocations involving TFE3, is a rare variant of renal cell carcinoma (RCC) [1].Unlike other more common subtypes of RCC, TFE3-rRCC has a distinct demographic profile, with a younger age at diagnosis, an advanced stage at presentation, and a female predominance [2].Due to the rarity of TFE3-rRCC, the understanding of the underlying mechanisms is currently poor, and the clinical treatment strategy in TFE3-rRCC is also largely undefined.To date, more than twenty TFE3 gene fusion partners have been identified, including ASPSCR1, SFPQ, NONO, PRCC, RBM10, MED15, etc.The diversity of fusion partners and chromosome structures affects biological behaviors and leads to the high heterogeneity of TFE3-rRCC, both morphologically and clinically [3].In clinical practice, diagnosis with TFE3-rRCC is mainly depended on IHC staining and FISH with break-apart probes, losing exact information about fusion partner.What's worse, being lack of effective therapy, patients with metastases had to be treated with established standard therapies developed for clear cell RCC (ccRCC), including tyrosine kinase inhibitors (TKI), mammalian target of rapamycin inhibitors (mTORi) and immune checkpoint inhibitors (ICI).So far, although there exists case reports and retrospective studies reporting some responses to these therapies, outcomes have been variable between series [4][5][6][7].Hence, the optimal therapy for TFE3-rRCC, especially with different fusion partner genes, remains to be determined.
Based on our previous transcriptomic analysis, TFE3-rRCC could be classified into 5 molecular clusters, suggesting that patients with TFE3-rRCC theoretically needed to be differentially treated [1].Nevertheless, no studies have yet compared responses of patients based on different fusion partners to systemic treatments.Therefore, we performed a single-center retrospective analysis in metastatic TFE3-rRCC patients with definite fusion partner genes based on RNA-seq, evaluating and comparing the efficacy of different systemic treatments and further exploring the molecular correlates.
Within the cohort, 65.8% (25/38) patients had metachronous metastases.Twenty-four (63.2%) patients developed metastasis in more than one organ.Involvement of retroperitoneal lymph node was the most common site of metastasis at the time of diagnosis of metastatic disease (60.5%).Lung, bone, liver and distal lymph node metastases were each observed at diagnosis of metastatic disease in 28.9%, 21.1%, 15.8%, 13.2% of cases, respectively.Among 32 patients with complete clinical data to evaluate IMDC risk, 78.1% (25/32) patients were stratified into intermediate/poor-risk group.
Univariate and multivariate cox regression analysis was further performed to identify prognostic factors that can determine the response to ICI.In the univariate analysis, we found that metastatic status (synchronous vs. metachronous), involvement of bone metastasis and ASPSCR1-TFE3 fusion were significantly associated with treatment outcomes (Suppl.Table 3).Kaplan-Meier curves demonstrated that patients with ASPSCR1-TFE3 fusion had longer PFS than those with other fusions when receiving ICI based combination therapy at firstline (median PFS: not reached vs. 6.5 months, P = 0.01) (Fig. 1E, Suppl.Table 4).In the contrary, patients with ASPSCR1-TFE3 fusion could not benefit from first-line therapy without ICI (median PFS: 3.0 vs. 5.1 months, P = 0.125) (Fig. 1F, Suppl.Table 4).In the multivariate analysis, only ASPSCR1-TFE3 fusion remained as the only independent prognostic factor for PFS in patients receiving first-line ICI based combination therapy (Suppl.Table 3).Regarding tumor response, ORR was 62.5% (5/8) in ASPSCR1-TFE3 rRCC patients, much higher than that in other TFE3-rRCC subtypes (10.0%, 1/10) (P = 0.019) (Fig. 1G, Suppl.Table 4).We also observed that patients with ASPSCR1-TFE3 fusion experienced longer OS compared to those with other fusions when receiving ICI based combination therapy at first-line or subsequent line.However, the difference did not reach statistical significance.(Suppl.Figure 4 A, 4B).Meanwhile, we have utilized data from the IMmotion151 cohort to corroborate our findings (Suppl.Table 5).Although the cohort included only 5 patients with metastatic TFE3-rRCC received ICI plus TKI combination therapy in the IMmo-tion151 cohort, it is notable that the sole responder (PR) was an ASPSCR1-TFE3 rRCC case, which also exhibited the best PFS.This outcome provides some validation of our findings.

Transcriptome features of ASPSCR1-TFE3 rRCC correlated with response to systemic treatment
We then attempted to figure out why patients with ASPSCR1-TFE3 fusion exhibited better response to ICI based combination therapy than other fusions through transcriptome analysis.RNA-seq was performed on 32 tumors, of which 31 had information of fusion partners.Additionally, we derived data from the IMmotion151 trial to validate findings from our cohort, and partner genes were identified in 10 out of 12 patients with TFE3 fusion (Suppl.Table 5) [8].
At the same time, in ASPSCR1-TFE3 rRCC, we also found enrichment of pathways involved in extracellular matrix (ECM) and collagen by GSEA (Fig. 2G, Suppl.Figure 6).Conversely, down-regulation of these pathways were observed in responders (PR), compared to non-responders (SD + PD), in all the patients who received first-line therapy without ICI, irrespectively of fusion types (Fig. 2H).Moreover, high expression of several collagen-related genes indicated shorter PFS in all the patients receiving therapy without ICI (Fig. 2I).These results demonstrated that ECM and collagenrelated pathways enriched in ASPSCR1-TFE3 rRCC might interfere with the potential efficacy of anti-angiogenic therapy.To our interest, positive correlations were observed between collagen-related genes, ECM and collagen-related signaling activity and expression of immune checkpoints (CD274, PDCD1), indicating the application of ICI (Suppl.Figure 7).
The immune microenvironment between TFE3-rRCC with ASPSCR1-TFE3 and other fusion partners was then explored.GSEA and ssGSEA both indicated enrichment of multiple immune-related pathways in ASPSCR1-TFE3 rRCC, which was further verified by data from the IMmo-tion151 trial (Fig. 2A-C).Similarly, up-regulation of immune-related pathways were also observed in responders receiving ICI based combination therapy (Fig. 2D).In addition, we calculated and compared the infiltration level of immune cells between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC using CIBERSORT algorithm (Suppl.Figure 8 A).Our focus then shifted to T cells due to their vital roles in tumor immunity.We observed that T cell levels are comparable between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC (Suppl.Figure 8B).we also evaluated the expression of several immune checkpoint molecules, finding that the ASPSCR1-TFE3 rRCC exhibited lower CTLA4 expression level than the non-ASPSCR1-TFE3 rRCC (P = 0.014) (Suppl.Figure 9).The results indicated a relative more inflamed immune microenvironment in ASPSCR1-TFE3 rRCC, which might explain why these patients could benefit from ICI based combination therapy.

Discussion
TFE3-rRCC is a rare but highly heterogeneous RCC entity, of which the clinical treatment landscape is largely undefined currently.Thus, the exploration of effective treatments remains an unmet need for this lethal disease.To our knowledge, this study represents the largest cohort investigating treatment outcomes and molecular correlates of metastatic TFE3-rRCC.Stratified by fusion partners using RNA-seq, it is the first time for us to reveal the association of therapeutic efficacy with different fusion partners among metastatic TFE3-rRCC.Accurately speaking, the superior efficacy of first-line ICI based combination therapy was predominately observed in ASPSCR1-TFE3 rRCC but almost not in non-ASP-SCR1-TFE3 rRCC.Analysis with transcriptomic data further revealed that enrichment of ECM and collagenrelated signaling might be involved in interfering with the potential efficacy of anti-angiogenic therapy.However, exclusively enrichment of angiogenesis and immune activities within ASPSCR1-TFE3 rRCC promised the better clinical outcomes with ICI plus TKI combination therapy.
The lack of standard treatment for patients with metastatic TFE3-rRCC is due mainly to its rarity, and the poor understanding of its underlying mechanisms.Early in 2021, we performed unsupervised transcriptomic analysis of 63 untreated TFE3-rRCCs and revealed five molecular clusters with distinct transcriptomic landscape [1].Surprisingly, tumors with ASPSCR1-TFE3 fusion were exclusively classified together, exhibiting high expression of angiogenesis/stroma/proliferation gene signatures.In contrast to ASPSCR1-TFE3 rRCC, non-ASPSCR1-TFE3 rRCC was featured by decreased angiogenesis gene (See figure on previous page.)Fig. 1 Characteristics and treatment outcomes of patients with metastatic TFE3-rRCC.A Heatmap showing clinicopathologic characteristics of patients with metastatic TFE3-rRCC patients (n = 38).B Swimmer plot showing the treatment response and duration of each patient receiving systemic treatments.C First-line PFS between patients receiving ICI based combination therapy and therapy without ICI at first-line.D First-line PFS between ASPSCR1-TFE3 rRCC patients receiving ICI based combination therapy and therapy without ICI at first-line.E First-line PFS between patients with ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC when receiving first-line ICI based combination therapy.F First-line PFS between patients with ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC when receiving first-line therapy without ICI.G Tumor response between patients with ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC when receiving first-line ICI based combination therapy signatures.Differential transcriptomic features among TFE3-rRCC with various fusion partners implied diversity with therapeutic strategies to substantially improve clinical outcomes.While almost all of studies with rRCC did not distinguish every fusion partner from each other.Efficacy evaluation could not get a definite conclusion based on types of fusion partner.Moreover, ICI plus TKI was mostly used as subsequent line treatment in these studies, while its therapeutic value as first-line treatment of TFE3-rRCC remains unknown.In our study, we initially observed the superior efficacy of first-line ICI based combination therapy in patients with ASPSCR1-TFE3 rRCC.
Totally speaking, omics analysis for the whole TFE3-rRCC demonstrated inactivation of angiogenesis, and several clinical explorations also confirmed the limited efficacy with TKI monotherapy [1].While comparing the transcriptome features of different fusions, ASPSCR1-TFE3 had higher enrichment of angiogenetic activity than other fusion partners, suggesting potential better response to anti-angiogenetic therapy.Unfortunately, in the present study, we still did not observe favorable response as we expected.To our interest, we observed up-regulation of ECM and collagen pathways in nonresponders receiving therapy with ICI.ECM network is established by multiple components, among which collagen plays the main role.Studies have demonstrated that there exists interplay between ECM remodeling and angiogenesis, for that ECM creates suitable environment that promotes endothelial cell proliferation, migration, and ultimately, angiogenesis [9].Nguyen et al. previously reported that ECM could lead to sorafenib resistance of hepatocellular carcinoma by activation of integrin-JNK signaling [10].Besides, increased ECM stiffness in liver metastases of colorectal cancer promoted resistance to bevacizumab [11].In our study, we demonstrated enrichment of ECM and collagen-related pathways in ASPSCR1-TFE3 rRCC, which might be the reason why this fusion subtype responded poorly to anti-angiogenic therapy, despite higher angiogenesis activity.In addition, ECM and collagen have vital immune modulatory functions within the tumor microenvironment [12].We also found that ECM and collagen-related signaling correlated positively with expression level of immune checkpoints, suggesting the potential application of ICI.
Aberrant angiogenesis is a hallmark of cancer, and contributes to the immune evasion of solid tumors [13,14].Hence, targeting the tumor vasculature to induce vessel normalization can provide a promising strategy to optimize the efficacy of immunotherapy [15].Previous studies have shown limited therapeutic outcomes of ICI monotherapy or doublet ICI combination therapy in rRCC [5,6].In our study, we observed favorable efficacy of ICI plus TKI only in ASPSCR1-TFE3 rRCC, probably through the synergistic effect between anti-angiogenic agents and immunotherapy.Moreover, compared to non-ASPSCR1-TFE3 rRCC, increased immune-related signaling were identified in ASPSCR1-TFE3 rRCC, which indicated a more inflamed immune microenvironment, and might further explain the efficacy of ICI based combination therapy.Given that other TFE3-rRCC subtypes responded poorly to ICI, further researches should be performed to explore the molecular mechanisms of these subtypes, and to propose pointed therapeutic strategy, accordingly.These findings mentioned above undoubtedly implied the importance of identifying fusion partner for every TFE3-rRCC patients in clinics.
This study has several limitations.Despite being the largest retrospective cohort of metastatic TFE3-rRCC, the small sample size due to the rarity of TFE3-rRCC may still limit the interpretation of the findings.Additionally, this was a single-center study thus selection bias is unavoidable.Further multi-center studies with larger sizes are required to shed further light on the development of effective therapies and selection strategies for patients suffering from this rare tumor.

Conclusion
In conclusion, the current study represents the largest cohort comparing treatment outcomes and investigating molecular correlates of metastatic TFE3-rRCC.By combination of clinical data and omics analysis, we demonstrated that ICI based combination therapy was an effective first-line treatment option for metastatic ASP-SCR1-TFE3 rRCC patients.Regarding with other fusion subtypes, further studies should be performed to explore (See figure on previous page.)Fig. 2 Transcriptomic landscape between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC.A Differentially expressed genes between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC.B Principal component analysis for the expression profiles between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC.C Enrichment of angiogenesis signaling pathways in ASPSCR1-TFE3 rRCC revealed by gene set enrichment analysis (GSEA).D Scores of angiogenesis signaling pathways between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3-rRCC by single sample GSEA (ssGSEA).E The mRNA expression level of VEGFA between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC.F Enrichment of VEGF signaling pathway in ASPSCR1-TFE3 rRCC revealed by GSEA.G Enrichment of ECM and collagen-related pathways in ASPSCR1-TFE3 rRCC revealed by GSEA.H Down-regulation of ECM and collagen-related pathways in responders of all patients receiving first-line therapy without ICI, irrespectively of fusion type.I First-line PFS between patients with high and low expression of collagenrelated genes when receiving first-line therapy without ICI, irrespectively of fusion type.J Enrichment of immune-related signaling pathways in ASPSCR1-TFE3 rRCC revealed by GSEA.K Scores of immune-related signaling pathways between ASPSCR1-TFE3 rRCC and non-ASPSCR1-TFE3 rRCC by ssGSEA.L Enrichment of immune-related signaling pathways in ASPSCR1-TFE3 rRCC revealed by GSEA in IMmotion151 cohort.M Enrichment of immune-related signaling pathways in responders receiving first-line ICI based combination therapy the molecular mechanisms, in order to propose pointed therapeutic strategy accordingly.